Chronic neuropathic pain is a common and debilitating ailment affecting more than 5 million people in the US, that dramatically affects the emotional and physical health of patients suffering from it. Opioids have only marginal success in treating chronic pain conditions and are limited by the development of tolerance. This proposal seeks to elucidate a novel mechanism of opioid tolerance and provide a new target for therapy, which could have the potential of aiding a large, underserved patient population. Microglia, the resident immune cell of the CNS have been shown to play an important role in both chronic pain and opioid tolerance. Similarly P2X receptors, which are activated by ATP/ADP, have been shown to mediate acute nociception and chronic pain states. We propose to study the role of microglial cells in morphine tolerance through the investigation of the following hypothesis: Morphine binds microglial mu opioid receptors, enhancing expression of P2X4 receptors, activating them and inducing cell migration. This activation and induction of cell migration leads to locally elevated cytokines and diffusible proinflammatory factors, inducing dorsal horn neuron sensitization and tolerance formation. We will investigate this hypothesis through the completion of the following Specific Aims: 1) Investigate the effect of mu opioid mediated enhancement of P2X4 receptor expression and microglial activation on cell migration in vitro and 2) Examine the relationship between P2X4 receptor activation and morphine tolerance in vivo. To investigate these Specific Aims, we will employ primary microglial cell culture and adult rats. Specific Aim 1 will be completed through the in vitro study of the effects of mu opioid agonists, antagonists and antagonists to P2X4 receptors on microglial migration. In a separate set of experiments, we will evaluate whether morphine induces microglial cells to increase P2X4 receptor expression. Specific Aim 2 will be completed through the in vivo study of the effects of P2X4 receptor antagonists on the development and maintenance of morphine tolerance and glial activation. The study of the effect of morphine on P2X4 receptor expression and activation has the potential to uncover a new mechanism of morphine tolerance. This interaction can be investigated to further understand why opioids such as morphine are ineffective in relieving pain in some patient populations and loose effectiveness over time in others. The goal of this research is to provide targets for new therapeutics that do not share the same propensity for tolerance formation and similar side effects as current opioids.